Magnetically templated tissue engineering scaffolds and methods of making and using the magnetically templated tissue engineering scaffolds

ABSTRACT

The present disclosure provides magnetically templated tissue scaffolds, methods of making the magnetically templated tissue scaffolds, and various methods of employing the scaffolds for tissue growth and repair in vitro and in vivo, including peripheral nerve repair.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application titled“Magnetically Templated Tissue Engineering Scaffolds and Methods ofMaking and Using the Magnetically Templated Tissue EngineeringScaffolds,” having Ser. No. 62/160,202, filed on May 12, 2015, which isentirely incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under grant number1R21NS093239 awarded by the National Institutes Health. The Governmenthas certain rights in this invention

BACKGROUND

Peripheral nerve injuries cause significant socioeconomic impact,resulting in over 8 million restricted activity days and over 5 milliondisability days per year. Over 200,000 peripheral nerve injury (PNI)repair procedures are performed each year in the US alone, with anestimated market for transected peripheral nerve injury repair of about$1.32-$1.93 billion in the US. Autograft remains the gold standardapproach for repairing injuries with gaps greater than 2 cm, commonlyfrom the patient's sural nerve. However, autografts result insignificant morbidity and functional deficit at the donor site, theiravailability is limited, particularly for extensive lesions, andmatching size of donor nerve to repaired nerve is often difficult.Furthermore, studies indicate motor function recovery occurs in only40-50% of patients.

Alternative therapies involving processed nerve allografts obtained fromdecellularized cadaveric nerve tissue were developed. These studies weretranslated into the Avance® graft, which has achieved functionalregeneration of nerve gaps up to 5 cm. Unfortunately, production ofdecellularized nerve allografts is limited by access to cadaveric tissueand is very costly because of the tedious and personnel-intensiveprocedure to clear the harvested nerve of undesired fat and connectivetissue. In addition, as with any allograft, there is some remote chanceof disease transmission. Other approaches, such as clinically availableartificial nerve guides (e.g., Neuragen®) and other technologies underdevelopment have been unsuccessful in repairing transected PNI with gapslonger than 2 cm. Reported tissue engineering scaffolds for peripheralnerve injury repair that lack aligned channels have failed to meet thesuccess of autografts for injuries greater than 20 mm.

SUMMARY

Briefly described, embodiments of the present disclosure provide abiocompatible tissue scaffold having aligned microchannels, methods ofmaking magnetically templated tissue engineering scaffolds, and methodsof repairing peripheral nerve damage.

An embodiment of the present disclosure provides a biocompatible tissuescaffold including a three-dimensional (3D) biocompatible scaffoldmaterial and a plurality of magnetically templated aligned microchannelshaving a diameter (e.g. about 1 μm to about 100 μm), wherein a portionof the microchannels or a network of interconnected microchannels, orboth, extend the length of the scaffold.

Embodiments of methods for making a biocompatible, tissue scaffoldhaving aligned microchannels include providing a biocompatible precursormaterial capable of polymerizing or crosslinking to form a gel or solidmaterial upon activation; providing microparticles comprising one ormore magnetic nanoparticles encapsulated in a dissolvable, biocompatiblematrix material; combining the microparticles and the biocompatibleliquid material in a mold; applying a magnetic field to the combinedmicroparticles and biocompatible liquid such that the microparticlesspatially align within the biocompatible material forming a plurality ofcolumns of adjacent microparticles, where the columns are substantiallydirectionally aligned with one another; activating the biocompatibleprecursor material to crosslink or polymerize such that thebiocompatible material substantially solidifies, forming a threedimensional (3D) scaffold around the aligned microparticles; dissolvingthe matrix material of the microparticles to produce a plurality ofaligned voids and microchannels within the scaffold; and allowing thedissolved matrix material and the released magnetic nanoparticles todiffuse out of the aligned voids and microchannels within the scaffold.

An embodiment of the present disclosure provides a biocompatible tissuescaffold having aligned microchannels formed by the methods of thepresent disclosure.

An embodiment of the present disclosure also provides methods ofrepairing peripheral nerve damage, which includes repairing a peripheralnerve gap with a biocompatible, magnetically templated tissue scaffoldas described herein or a biocompatible tissue scaffold made by themethod as described herein.

Other methods, compositions, features, and advantages of the presentdisclosure will be or become apparent to one with skill in the art uponexamination of the following drawings and detailed description. It isintended that all such additional compositions, methods, features, andadvantages be included within this description, and be within the scopeof the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with referenceto the following drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIG. 1 is a schematic illustration of an embodiment of a method of thepresent disclosure for using magnetic microparticles for makingmicrochannels in a scaffolding material to provide templated tissuescaffolds.

FIGS. 2A-2C illustrate magnetic microparticles (FIG. 2A (optical) andFIG. 2B (SEM)) and formation of columnar structures of themicroparticles in a scaffolding precursor material in a magnetic field(FIG. 2C, approximately 50 mT).

FIGS. 3A-3B illustrate use of a ring magnet to align microparticles in ascaffold precursor material (e.g., pre-hydrogel mixture) in a mold (FIG.3A) and a formed hydrogel containing magnetic microparticles (slicedlongitudinally) obtained from a the cylindrical mold in FIG. 3A aftercrosslinking.

FIGS. 4A-4B illustrate a crosslinked hydrogel of approximately 10 mm inlength, showing a columnar magnetic microparticle structure beforedissolution of the particles (FIG. 4A) and the microchannels remainingafter dissolution of the microparticles in EDTA (FIG. 4B).

FIG. 5 illustrates neurite ingrowth of GFP+P1 rat dorsal root ganglia(DRG) on templated HA+collagen I hydrogel, demonstrating the ability tocreate templated tissue scaffolds to support cell attachment and growth.

FIGS. 6A-6C illustrate aligned commercial microparticles in water (FIG.6A), GMHA pre-gel solution (FIG. 6B), and GM HA hydrogel (FIG. 6C).

FIGS. 7A-7C illustrate aligned alginate microparticles in water (FIG. 7a), GMHA pre-gel solution (FIG. 7B), and GMHA hydrogel (FIG. 7C).

FIGS. 8A-8C illustrate composite images of crosslinked GMHA hydrogelswith unaligned microparticles (FIG. 8A), aligned microparticles (FIG.8B), and aligned and degraded microparticles (FIG. 8C).

FIG. 9 Illustrates porous channels remaining after particle degradation.

FIGS. 10A-10B illustrate templated (FIG. 10A) and non-templated (FIG.10B) hydrogels after 2 weeks of implantation in a rat sciatic nervedefect.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit (unlessthe context clearly dictates otherwise), between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification that areincorporated by reference, as noted in the application, are incorporatedby reference to disclose and describe the methods and/or materials inconnection with which the publications are cited. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present disclosure is notentitled to antedate such publication by virtue of prior disclosure.Further, the dates of publication provided could be different from theactual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of, chemistry, organic chemistry, organometallicchemistry, polymer chemistry, microbiology, tissue engineering, and thelike, which are within the skill of the art. Such techniques areexplained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a cell” includes a plurality of cells. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise. In this disclosure, “consisting essentiallyof” or “consists essentially” or the like, when applied to methods andcompositions encompassed by the present disclosure refers tocompositions like those disclosed herein, but which may containadditional structural groups, composition components or method steps (oranalogs or derivatives thereof as discussed above). Such additionalstructural groups, composition components or method steps, etc.,however, do not materially affect the basic and novel characteristic(s)of the compositions or methods, compared to those of the correspondingcompositions or methods disclosed herein. “Consisting essentially of” or“consists essentially” or the like, when applied to methods andcompositions encompassed by the present disclosure have the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

Definitions

In describing and claiming the disclosed subject matter, the followingterminology will be used in accordance with the definitions set forthbelow.

As used herein, the term “engineered” indicates that the engineeredobject is created and/or altered by man. An engineered object mayinclude naturally derived substances, but the object itself is alteredin some way by human intervention and design.

As used herein the term “channels” or “microchannels” refers to tubulesor tube-like formations within a construct. The channels have agenerally elongated and/or cylindrical shape, with a generally circularcross-section. The channels have an open (e.g., hollow or substantiallyhollow) interior (referred to herein as a “lumen”) creating avia/conduit that forms a scaffold/template for the growth ofcells/tissues and can also facilitate the movement of fluid, cells, andother materials within and/or through the construct. In embodiments, themicrochannels can have a diameter in the micron range (e.g., 1-100 μm,including diameters from 5-20 μm, and diameters with an average of about10 μm).

As used herein the term “biocompatible” refers to the ability toco-exist with a living biological substance and/or biological system(e.g., a cell, cellular components, living tissue, organ, etc.) withoutexerting undue stress, toxicity, or adverse effects on the biologicalsubstance or system.

The term “biocompatible scaffold material” refers to any compoundsubstance with sufficient structural stability to provide a substrate tosupport the growth of a living biological substance (e.g., livingcells). In embodiments of the present disclosure the biocompatiblescaffold material has a three-dimensional structure (rather than aplanar, 2-dimensional structure) to support three-dimensional growth ofliving cells. In embodiments, the biocompatible scaffold material ismade from a liquid/semi-liquid material that can be crosslinked and/orpolymerized into a matrix that provides a more solid (e.g., solid, gel,semi-solid, etc.) scaffold.

The term “matrix material” refers to several different types ofsemi-solid to solid materials with a gel-like and/or solid consistencyand a structure capable of supporting the growth of living biologicalsubstances (e.g., living cells). Both synthetic and naturally derivedgel matrix materials exist and are in use by those of skill in the art.Gel matrix materials include hydrogels, such as biocompatible naturallyderived or synthetic hydrogels, such as, but not limited topolymer-based hydrogels, PEG based hydrogels, alginate, cellulose,keratin, elastin, collagen, and the like. Gel matrix materials alsoinclude biocompatible polymer or copolymer based gel materials, such aspolymer and copolymer based hydrogels. Gel matrix materials may alsoinclude a gelling agent or crosslinking agent (e.g., formaldehyde,glutaraldehyde, etc.) to increase the structural stability of the gel(e.g., to give it more “solid” characteristics).

As used herein, the term “solid” shall include “semi-solid” materials,and “liquid” shall include “semi-liquid” materials.

The term “polymer” includes any compound that is made up of two or moremonomeric units covalently bonded to each other, where the monomericunits may be the same or different, such that the polymer may be ahomopolymer or a heteropolymer. Representative polymers includepolyamides, such as polypeptides, poly-N-substituted glycines(polypeptoids), polysaccharides, polyethylene glycol or polyethyleneoxide, plastics (e.g., poly-L-lactic acid, poly-L-glutamic acid andco-polymers thereof), nucleic acids and the like, where the polymers maybe naturally occurring, non-naturally occurring, or synthetic. The term“bio-polymer” refers to a polymer made of biologically-derived and/orbiologically compatible compounds

The term “attached” or the phrases “interacts with” and “associatedwith” refers to a stable physical, biological, biochemical, and/orchemical association. In general, association can be chemical bonding(e.g., covalently or ionically), a biological interaction, a biochemicalinteraction, and in some instances a physical interaction. Theassociation can be a covalent bond, a non-covalent bond, an ionic bond,a metal ion chelation interaction, as well as moieties being linkedthrough interactions such as, but not limited to, hydrophobicinteractions, hydrophilic interactions such as hydrogel bonding,charge-charge interactions, π-stacking interactions, combinationsthereof, and like interactions.

Use of the phrase “biomolecule” is intended to encompassdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleotides,oligonucleotides, nucleosides, proteins, peptides, polypeptides,selenoproteins, antibodies, protein complexes, peptide nucleic acids,combinations thereof, and the like. In particular, the biomolecule caninclude, but is not limited to, naturally occurring substances such aspolypeptides, polynucleotides, lipids, fatty acids, glycoproteins,carbohydrates, fatty acids, fatty esters, macromolecular polypeptidecomplexes, vitamins, co-factors, whole cells, eukaryotic cells,prokaryotic cells, microorganisms, or combinations thereof.

The phrase “bioactive agent” includes a biomolecule or otherbiocompatible compound that has some activity, use, and/or effect in abiological system or in relation to another biomolecule.

The terms “polypeptide” and “protein” as used herein refer to a polymerof amino acids of three or more amino acids in a serial array, linkedthrough peptide bonds. The term “polypeptide” includes proteins, proteinfragments, protein analogues, oligopeptides and the like. The term“polypeptides” contemplates polypeptides as defined above that areencoded by nucleic acids, produced through recombinant technology(isolated from an appropriate source such as a bird), or synthesized.The term “polypeptides” further contemplates polypeptides as definedabove that include chemically modified amino acids or amino acidscovalently or non-covalently linked to labeling ligands.

DISCUSSION

In accordance with the purpose(s) of the present disclosure, as embodiedand broadly described herein, embodiments of the present disclosurerelate to magnetically templated tissue scaffolds, methods of making thescaffolds, and various methods of employing the scaffolds for tissuegrowth and repair in vitro and in vivo, peripheral nerve repair, andmany other uses.

Tissue engineering scaffolds with aligned pores and channels are ofinterest in a variety of biomedical applications, including nerve injuryrepair. As discussed above, due to limitations in existing technologiesfor repair of peripheral nerve injury (PNI), a need exists for abioengineered peripheral nerve scaffold that includes the architectureor both chemical and architectural components of natural peripheralnerve tissue to facilitate repair of any size nerve gaps, includinglonger nerve gaps, without the disadvantages of autographs, allographs,and existing nerve guide conduits. It is believed that the success ofdecellularized nerve allografts in repairing 2-5 cm gaps stems, at leastin part, from preservation of the native basal lamina extracellularmatrix (ECM) microstructure of the nerve. Specifically, preservation ofapproximately 10 μm diameter basal lamina tubes helps direct axonalgrowth and guide nerve reconnection. However, the fact thatdecellularized nerve allografts fail in repairing PNI with 5-12 cm gaps,for which autografts are successful, indicates that control overtopography alone faces limitations in repair of long PNI gaps.Processing conditions used to obtain decellularized nerve allograftsremove all chemical and biological cues that may aid in directing axonalgrowth, and incorporating these chemical and biological cues aposteriori has been challenging, particularly for long grafts. This maybe a contributor to failure of nerve allografts in repairing gapsbetween about 5-12 cm. Thus, a need exists for regeneration scaffoldswith tubular microstructure that mimics that of natural nerve, includingaligned, gap-spanning tubes with a diameter comparable to naturalperipheral nerve (e.g., approximately 10 μm) embedded in a biocompatiblematrix and obtained through methods that are compatible withincorporating chemical and biological cues.

Magnetic fields to control nano- and microtopography, or to directassembly of cells or tissue engineering scaffolds can be used in variousapplications. Alignment of collagen and fibrin fibers through magneticfields has been studied to direct cell growth, but suffers practicallimitations for PNI repair because fiber alignment relaxes once themagnetic field is removed. Magnetic alignment of anisotropic dissolvableparticles has been used to create bone cement scaffolds with anisotropicporosity, but the reported materials lack the gap-spanning alignedmicrochannels required for PNI repair applications. Magnetic hydrogelshave been reported that respond to magnetic fields because of magneticnanoparticles retained within the hydrogel, but lack the tubularfeatures required for PNI repair. Magnetic fields have also been used todirect 2D patterning of dissolvable magnetic sugar particles, leavingbehind a scaffold with patterned 2D porosity, but the pores are toolarge to effectively direct axon growth and do not form continuoustubular guidance conduits that span dimensions relevant for PNI repair.Magnetic fields have also been used to direct assembly of cells andhydrogels, but not with the topographic features, nor at the size scalesneeded for PNI repair. The present disclosure provides magnetictemplating distinct from the other approaches in its use of magneticmicrospheres that form aligned, gap-spanning columnar structures andthat, once dissolved, leave behind a tubular microstructure withdimensions suited for effective PNI repair.

The present disclosure provides a new approach to provide templatedtissue scaffolds with controllable diameter and length, with alignedpores and microchannels to provide appropriate structure for growth oftissue, such as nerve tissue suitable for peripheral nerve repair.Briefly described, in embodiments, a magnetic field is applied to a moldcontaining a crosslinkable and/or polymerizable biocompatible precursormaterial (e.g. a biocompatible, polymerizable polymer, a crosslinkableor photopolymerizable hydrogel of naturally derived biomaterial, etc.)mixed with a plurality of sacrificial magnetic microparticles (e.g.,microparticles of a sacrificial/dissolvable matrix material withencapsulated/embedded magnetic nanoparticles). The magnetic field causesthe microparticles to align and form a plurality of lines/columns ofadjacent microparticles, where the columns are also substantiallyaligned with each other (e.g., substantially oriented in the samedirection, substantially parallel, etc.). In embodiments, the magneticfield is applied such that the microparticles substantially align toform columns along the length (e.g., greater dimension) of the mold(e.g., longitudinal rather than crosswise alignment). After allowing theparticles to align, while still applying the magnetic field orimmediately after removal of the magnetic field, appropriate stimulus isapplied to the crosslinkable/polymerizable biocompatible material toactivate the crosslinking/polymerization of the material (e.g.,application of UV light for photopolymerizable materials, addition of achemical crosslinker, heat activation, etc.). In embodiments thecrosslinking/polymerization is done while still applying the magneticfield to ensure the magnetic microparticles remain in columns within thebiocompatible precursor material. In some embodiments, thecrosslinking/polymerization is done immediately after removing thematerial from the magnetic field such that the materialpolymerizes/crosslinks before the microparticle columns/chainsdisassemble (e.g., when the precursor material is somewhat viscous suchthat the microparticles do not diffuse out of alignment immediately).

Upon crosslinking/polymerizing of the material, the biocompatiblematerial substantially solidifies (gel, solid/semi-solid) to form athree dimensional (3D) scaffold around the aligned microparticles. Afterformation of the scaffold, the matrix material of the microparticles isdissolved/sacrificed, and the dissolved material and the magneticnanoparticles diffuse/leach out of the scaffold through themicrochannels and pores left behind by the microparticles. Inembodiments, additional biomolecules (e.g., cells, proteins,carbohydrates, nucleic acids, etc.) may be included in the biocompatiblescaffold material and/or matrix material of the microparticles, and allof the polymerizing/crosslinking/dissolution steps are carried out inbiocompatible conditions that are non-toxic/non-harmful to any suchbiomolecules (e.g., they do not interfere with the intendedpurpose/activity of the biomolecules).

One of the principal functions of a biological scaffold is to directcell behavior such as migration, proliferation, differentiation,maintenance of phenotype, etc. by facilitating sensing and responding tothe environment via cell-matrix and cell-cell communications. Inembodiments, the present disclosure provides crosslinked or polymerizedscaffolds of biocompatible and/or naturally derived biomaterialscontaining aligned tubular microstructure that mimics natural nervetissue through magnetic templating as shown in the embodimentillustrated in FIG. 1. With magnetic templating, the length, diameter,connectivity, and areal density of microchannels remaining afterdissolution of magnetic microparticles can be tuned through control ofmicroparticle concentration, diameter, and magnetic nanoparticlecontent, and through magnetic field conditions. Furthermore, theappropriate magnetic fields can be generated to obtain scaffolds thatare ˜12 cm long while permitting crosslinking of the hydrogel. Finally,choosing microparticle materials with mild dissolution conditions thatare biocompatible allows incorporation of chemical and biological cuesinto the scaffold before or after templating.

In embodiments of the present disclosure, the scaffold material caninclude a matrix of a synthetic or naturally derived resorbable ornonresorbable material, where the matrix is prepared by crosslinking orpolymerization of a biocompatible precursor matrix-forming material. Inembodiments, the scaffold material is both biocompatible andbiodegradable. In embodiments, the precursor material is initially in aliquid or semi-liquid state before crosslinking/polymerization so thatthe precursor material has sufficient fluid properties to allow movementof the microparticles within the precursor material prior tocrosslinking/polymerization. The crosslinking/polymerization isperformed in the presence of a collection of dissolvable magneticmicroparticles and under the application of a magnetic field, orimmediately after, which causes the alignment of the magnetic particlesin aligned structures. In embodiments, the dissolvable magneticmicroparticles have sizes in the range of about 100 nm to 100 μm. Forsome applications the magnetic microparticles have sizes in a range ofabout 10 to 50 microns. In other embodiments, the particles have sizesin intermediate ranges within the 100 nm to 100 μm range, depending onthe desired diameter of the microchannels formed once the alignedmicroparticles have dissolved and diffused away. After crosslinking orpolymerization of the scaffold matrix material, the scaffold is treatedin such a way to dissolve the magnetic particles, removing theirconstituents and leaving empty voids and channels in the scaffold. Thesevoids and channels may then direct growth, including directional growth,of cells in the scaffold. The voids and channels may also be modifiedduring or post-preparation with cell adhesion factors and otherdesirable biomolecules. In embodiments, the voids and channels arealigned along a direction of the scaffold over a length of about 20 mmor more.

The magnetically templated regeneration scaffolds of the presentdisclosure may replace clinical use of processed nerve allografts andnerve autografts for 2-12 cm PNI gaps. Regeneration scaffolds withtubular microstructure and that can span gaps up to 5 cm, could repairgaps without the need of chemical and biological cues and couldpotentially replace processed nerve allografts, which successfullyrepair gaps up to 5 cm without the need of chemical or biological cues.In embodiments, magnetically templated regeneration scaffolds of thepresent disclosure with aligned microchannels that span gaps up to 12 cmlong and incorporate chemical and biological cues can direct axon growthpast the apparent 5 cm limit of topography. Such regeneration scaffoldscould potentially eliminate the need of nerve autografts. Furthermore,the magnetically templated scaffolds of the present disclosure havepotential applications for tissue engineering in otherdiseases/conditions beyond PNI repair.

In embodiments of the magnetically templated scaffolds of the presentdisclosure, naturally derived biomaterials were selected as buildingblocks of the proposed scaffolds because of their biocompatibility andinherent role in wound healing. In addition, when using naturalscaffolds (e.g., extracellular matrix (ECM)-based scaffolds), there isless concern with immunogenicity; the body will inherently remodelnatural materials, unlike with many synthetic materials in which therecould be issues associated with toxicity and/or adverse effectsassociated with either the material itself or its degradation products.These features grant naturally derived biomaterials with potential fornear term clinical success. However, the magnetic templating methods ofthe present disclosure are also compatible with hydrogels and otherscaffolding materials made of synthetic biomaterials.

In an embodiment the biocompatible material for forming the scaffold isselected from a biocompatible, crosslinkable hydrogel. In embodiments,the biocompatible precursor material is in a liquid or semi-liquid formprior to crosslinking/polymerization into a gel/solid/semi-solid matrixthat forms the biocompatible scaffold. Examples of biocompatiblematerials that can be used to form the scaffold of the presentdisclosure include, but are not limited to, hyaluronic acid, collagen,polyethylene glycol, fibrin, and the like. In embodiments, the scaffoldmaterial is formed from a chemically crosslinked hyaluronic acidhydrogel. Hyaluronic acid has advantageous features, such as, but notlimited to: it is biodegradeable and biocompatible, it is FDA approved(e.g., for use in dermal fillers), it is a natural component ofextracellular matrix, it allows for incorporation of otherchemical/biological factors, and it forms an amorphous solid. In anotherembodiment the scaffold material consists of suitable crosslinkedcollagen, which also shares many of the same advantages. Otherbiocompatible hydrogels and other biocompatible material may be employedas the scaffolding material. Also, combinations of such materials canalso be used. For instance, in embodiments, the scaffolding material mayinclude both hyaluronic acid and collagen and/or other matrix-formingmaterials.

In embodiments, the biocompatible material for forming the scaffold isinitially in a liquid (including liquid and semi-liquid states) statebut is capable of being crosslinked or polymerized to form a matrixmaterial that is a gel, solid/semi-solid material upon activation (e.g.,chemically or physically, such as, but not limited to application of UVlight, application of heat, addition of a chemical crosslinker oractivator, etc.). Once crosslinked/polymerized, the biocompatiblematerial forms a matrix that provides a three-dimensional (3D) scaffold.In the embodiments of the present disclosure, this scaffold is formedaround the microparticles, which are present in the biocompatiblematerial prior to activation of the crosslinking/polymerization, suchthat the location of the microparticles leaves a void in the scaffoldupon removal of the microparticle. Since the microparticles can bealigned by application of a magnetic field and then dissolved afterscaffold formation, the adjacent voids created by the alignedmicroparticles form tubules or microchannels in the scaffold. Byapplication of the magnetic field, the directional alignment of themicroparticles can be controlled, such that in embodiments, themicrochannels are substantially aligned along the length of thescaffolding material. In embodiments, a portion of the microchannelsextend the full length of the scaffolding material. In embodiments, aportion of the microchannels extending the length of the scaffoldinghave an opening at each end of the scaffolding. In embodiments, thescaffolding material is formed in a mold to provide shape and support tothe biocompatible scaffolding precursor material prior tocrosslinking/polymerizing. The mold can be later removed. Inembodiments, the mold is a sacrificial material that is later removedusing known techniques.

In an embodiment of the templated tissue scaffold of the presentdisclosure and methods of making the scaffold, the microparticlesinclude magnetic nanoparticles encapsulated in a dissolvable matrix. Inembodiments, the dissolvable magnetic microparticles have sizes in therange of 100 nm to 100 μm. In an embodiment, the magnetic nanoparticlesinclude iron oxide nanoparticles. In an embodiment, the magneticnanoparticles are also combined with a surfactant, charged species, orpolymer that confers colloidal stability in aqueous media. Inembodiments, the dissolvable matrix material is a polymer matrixmaterial. In embodiments, the matrix material is a biocompatiblematerial such as, but not limited to, calcium alginate, a polyethyleneglycol based hydrogel that dissolves in response to a stimulus, and thelike. For embodiments where the matrix material is calcium alginate, thedissolution step may be performed using a sodium citrate, EDTA, and/oralginate-lyase solution. In an embodiment, the magnetic microparticlesinclude tetramethyl ammonium hydroxide stabilized iron oxidenanoparticles encapsulated in calcium alginate to form microparticlesthrough an emulsion crosslinking technique under optimized conditions toyield particles with diameters in the range of about 1-20 μm. In someembodiments, the magnetic particles have a mean diameter of about 10 μm.In embodiments, after activation and formation of the scaffoldingmaterial and dissolution of the microparticles, the formed microchannelshave a diameter of about 1 to about 20 μm. In embodiments, themicrochannels have an average diameter of about 10 μm. In someembodiments microfluidic devices can be used to create the alginatemicroparticles, which allows for size control in the 1-400 μm range. Forinstance, in embodiments, the magnetic microparticles includetetramethyl ammonium hydroxide stabilized iron oxide nanoparticlesencapsulated in calcium alginate to form microparticles throughmicrofluidic droplet formation using a flow focusing device, or othersuch devices suitable for making microdroplets.

In embodiments of the templated scaffolds and methods of the presentdisclosure, the synthetic or naturally derived biocompatible materialmay be resorbable or nonresorbable. In embodiments, the biocompatiblescaffolding material may be biodegradable such that the scaffoldingmaterial degrades in vivo or in vitro over time. In some embodiments,the biocompatible scaffolding may contain living cells or otherbiomolecules prior to crosslinking or polymerization in the presence ofmagnetically aligned nanoparticles. Such cells or biomolecules wouldthen be left behind in the scaffold material once the magneticmicroparticles are dissolved. Such embodiments can provide controlledrelease of active agents into the pores or microchannels left afterdissolution and leaching of the magnetic particles, providing biologicalcues that direct cell growth into the scaffold and phenotypedifferentiation within the scaffold.

Similarly, in some embodiments, the magnetic microparticles may includecells or biomolecules (such as, but no limited to, growth factors,enzymes, and the like) co-encapsulated with magnetic nanoparticles inthe dissolvable matrix material. Then, upon dissolution of the particlematrix, the cells or biomolecules are left behind in the resulting poresand channels of the scaffold. This may be useful for rapidly populatinglarge scaffolds with cells. It may also be useful for directinginvasion, growth, and differentiation of cells in the scaffolds. Oneexample application includes populating channels with Schwann cells,which may direct axonal growth into the scaffold. In embodiments,autologous cells from the recipient can be cultured and encapsulated inthe pre-crosslinked or pre-polymerized scaffolding material and/or inthe microparticle matrix as a way to seed the scaffold and/or themicrochannels with recipient's cells to stimulate growth. In someembodiments, the presence of cells in the magnetic microparticles and inthe scaffold matrix is combined.

The present disclosure includes templated scaffolds prepared by theprocesses including the methods of the present disclosure describedabove. Such magnetically templated tissue scaffolds include athree-dimensional, biocompatible, and optionally biodegradable,scaffolding material (e.g., a biocompatible hydrogel, a biocompatiblepolymer, etc.) that is formed from a precursor material that iscrosslinkable/polymerizable under biocompatible conditions. Inembodiments, magnetically templated tissue scaffolds of the presentdisclosure also include a plurality of magnetically templated alignedmicrochannels where a portion of the microchannels (as a singlemicrochannel or a group of interconnected microchannels) extend thelength of the scaffold such that, in combination, the connectedmicrochannels span the length of the scaffold. In embodiments, at leasta portion of the microchannels (single or interconnected) extend thelength of the scaffold with openings at each end of the scaffold. Inembodiments, the microchannels are at least substantially aligned witheach other and are substantially directionally aligned along the lengthof the tissue scaffold. In embodiments, the microchannels have adiameter of 1 to about 20 μm. In embodiments, the microchannels have anaverage diameter of about 10 μm. In embodiments, the scaffold alsoincludes living cells and/or other biomolecules within the scaffoldingmaterial and/or within the interior space (e.g., lumen) of themicrochannels. In embodiments, the scaffolds have a length of about 2-12cm, although in some embodiments, the scaffolds can have a lengthgreater than 12 cm or less than 2 cm. In embodiments, the scaffolds havea length of about 2-5 cm. In embodiments, they have a length of about5-12 cm. In embodiments, a least a portion of the microchannels withinthe scaffold extend the length of the scaffold and thus have a length(may be length of interconnected microchannels) between about 2-12 cm.In embodiments, a single microchannel spans the length of the scaffold,in other embodiments, some microchannels join with other microchannelsto jointly span the length of the scaffold.

Embodiments of the present disclosure also include methods of inducingcell growth in the biocompatible, magnetically templated scaffolds ofthe present disclosure in vivo or in vitro. Methods also include methodsof repairing peripheral nerve damage by using the biocompatible tissuescaffolds of the present disclosure to repair nerve gaps of about 2-12cm, such as in the embodiment described in Example 2, below. Smallernerve gap repair is also possible with the scaffolds and methods of thepresent disclosure, such as gaps smaller than 2 cm, e.g., from about 2mm to about 2 cm. Typically, for a gap less than 2 mm, it can besurgically repaired without the need for a scaffolding material orgraft.

Additional details regarding the methods and compositions of the presentdisclosure are provided in the Examples below. The specific examplesbelow are to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. Without furtherelaboration, it is believed that one skilled in the art can, based onthe description herein, utilize the present disclosure to its fullestextent. All publications recited herein are hereby incorporated byreference in their entirety.

It should be emphasized that the embodiments of the present disclosure,particularly, any “preferred” embodiments, are merely possible examplesof the implementations, merely set forth for a clear understanding ofthe principles of the disclosure. Many variations and modifications maybe made to the above-described embodiment(s) of the disclosure withoutdeparting substantially from the spirit and principles of thedisclosure. All such modifications and variations are intended to beincluded herein within the scope of this disclosure, and protected bythe following claims.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. In an embodiment, the term “about” can includetraditional rounding according to significant figures of the numericalvalue. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ toabout ‘y’”.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the examples describe some additional embodiments of thepresent disclosure. While embodiments of present disclosure aredescribed in connection with the examples and the corresponding text andfigures, there is no intent to limit embodiments of the presentdisclosure to this description. On the contrary, the intent is to coverall alternatives, modifications, and equivalents included within thespirit and scope of embodiments of the present disclosure.

Example 1 Magnetically Templated Hyaluronan Hydrogel Tissue Scaffolds

Tissue engineering scaffolds (scaffolds) with aligned pores and channelscan be useful for a variety of biomedical applications, including nerveinjury repair. This example describes an embodiment of engineeringscaffolds with aligned pore and channel structures through magneticallyguided assembly of dissolvable magnetic particles.

The present example describes making and testing of magneticallytemplated scaffolds using magnetic alginate microparticles (MAMs)including biocompatible iron oxide nanoparticles embedded in acrosslinked calcium alginate matrix. Iron oxide nanoparticles are onecomponent in various FDA approved magnetic resonance imaging contrastagents, and are believed to be biocompatible and bio-absorbable.Furthermore, in magnetic templating, the majority, if not all, of theiron oxide nanoparticles are removed during the MAM dissolution step.Alginate was selected as the dissolvable matrix for the MAMs due, atleast in part, to its biocompatibility, its use to encapsulate viablecells for culture, and because dissolution can be achieved under mildconditions that do not affect biomolecules or cell viability, such asdescribed in the following references, which are hereby incorporated byreference herein (W. R. Gombotz and S. F. Wee, “Protein release fromalginate matrices,” Advanced Drug Delivery Reviews, vol. 64, pp.194-205, 2012; H. H. Tønnesen and J. Karlsen, “Alginate in drug deliverysystems,” Drug development and industrial pharmacy, vol. 28, pp.621-630, 2002; and C. Bucke, “Cell Immobilization in Calcium Alginate,”Methods in enzymology, vol. 135, pp. 175-189, 1987). These qualities ofalginate encapsulated microparticles are compatible with embodimentswhere cells or biomolecules are incorporated into the scaffold ormicroparticles to provide chemical and biological cues.

In the present example, hyaluronan hydrogels containing collagen as thebase biomaterial were employed. Hyaluronan (HA) is a natural componentof adult brain and spinal cord ECM, is biocompatible, non-immunogenic,and is easy to modify. Collagen, while less prevalent in the adultbrain, has been successfully combined with HA to enhance cell adhesionproperties in nerve tissue repair applications. Both HA and collagenbiodegrade into harmless natural components. Due to these featuresHA/collagen hydrogels were selected as suitable material for thescaffolding matrix in the magnetically templated regeneration scaffolds.

To illustrate feasibility, MAMs were obtained by water-in-oil emulsioncrosslinking of sodium alginate and iron oxide nanoparticle mixturesusing calcium chloride. The iron oxide nanoparticles were synthesizedand peptized according to procedures described in Mérida, A. Chiu, A.Bohórquez, L. Maldonado, M.-E. Pérez, L. Pericchi, M. Torres-Lugo, C.Rinaldi, “Optimization of synthesis and peptization steps to obtain ironoxide nanoparticles with high specific absorption rates.” Journal ofMagnetism and Magnetic Materials, 394:361-371, 2015, which is herebyincorporated by reference herein. Briefly, aqueous solutions of iron(II) and iron (III) salts are prepared in degassed deionized water(total metal concentration of 0.3 M, with 2:1 Fe³⁺:Fe²⁺ molar ratio).These solutions are then heated and NH₄OH added, followed by one-hourreaction at elevated temperature (85° C.). The pH was kept between 8.0and 9 using NH₄OH. The resultant nanoparticle solution was cooled toroom temperature, centrifuged, and magnetically decanted. Peptizationwith tetramethylammonium hydroxide (TMAOH) was achieved using a IO/TMAOHvolume ratio of 2 by dispersing the iron oxide nanoparticles in thepeptizing agent followed by application of ultrasound (XL2020, MisonixInc.). The resulting nanoparticles were centrifuged and magneticallydecanted, dried overnight, and then suspended in water to yield aqueousnanoparticle solutions, which were used as stock solutions for furtheruse.

For emulsion MAM production, an aqueous phase of 20 mg/mL sodiumalginate and 10-200 mg/mL Iron Oxide Peptized Nanoparticles in H₂O wasadded dropwise to the continuous phase of mineral oil with 5% Span 80surfactant while shearing on a homogenizer for ˜10 minutes at 4000 RPM.10% CaCl solution was then added at a rate of 3 mL/min to crosslink thealginate, and homogenization was continued for 3 minutes for completemixing and alginate gelation. After homogenization, the solution wasadded to 100% EtOH, and the particles were separated from the continuousphase using either centrifugation or magnetic separation. Using eithermethod, the supernatant was discarded after separation, and theparticles were resuspended in 100% EtOH for at least 4 washes. Afterparticle purification, the particles were resuspended in DI H₂O andaliquoted into pre-weighted tubes for lyophilization to determine thedried weight of particles. Particles were stored in a desiccator at −20°C. and resuspended in water at known concentrations prior to use.Optical microscopy and scanning electron microscopy indicate these MAMswere irregular in shape and polydisperse in size, with diameters of upto 5 μm (FIGS. 2A and 2B).

MAMs are easily dispersed in HA/collagen pre-hydrogel mixtures andreadily align into long columnar structures upon application of amagnetic field (FIG. 2C). The length of these columnar structuresappears to be determined by the size of the magnet used and theconcentration of the particles in the pre-hydrogel mixture, andalignment for hydrogels >2 cm long was achieved using a cylinder magnet(FIGS. 3A &3B). The cylindrical magnet used in the present example was aRX08X0 magnet from K&J Magnetics. Using GMHA hydrogels, the maximumparticle concentration that can be added appears governed by the abilityof UV light to penetrate the hydrogel for GMHA crosslinking. Hydrogelgelation has been shown to occur up to MAM concentrations of 10-20mg/mL, dependent on MAM synthesis conditions, magnetic nanoparticleconcentration, average MAM diameter, and overall purity.

Columnar structures were preserved under the magnetic field duringcrosslinking of the HA/collagen hydrogel using UV (FIG. 4A), and theMAMs readily dissolve using the Ca²⁺ chelating agents (FIG. 4B).

In this study, EDTA was used to dissolve the MAMs. While EDTA iscompatible with the presence of some biomolecules, it may be replacedwith other dissolution compounds in embodiments that incorporate cellsinto the hydrogels. For example, MAMs can also be dissolved using sodiumcitrate, which is commonly used to dissolve alginate beads used toencapsulate cells in 3D cell culture. Alginate-lyase can be used toenzymatically degrade the alginate into smaller MW fragments for morerapid diffusion through the hydrogel matrix. Finally, preliminarystudies of in vitro ingrowth of rat dissociated dorsal root ganglia(DRG) into HA/collagen hydrogels with templated channels indicates thisbase biomaterial is suitable for scaffolds to direct axon growth (FIG.5).

Additional methods and templated tissue scaffolds were alsodemonstrated. A comparison of the results utilizing commercialmicroparticles and the alginate microparticles described above is shownin FIGS. 6A-6C and FIGS. 7A-7C. Aligned commercial microparticles (BangsLaboratories Inc., PMC3N, ProMag™ 3 μm diameter polymer-based magnetitespheres) are shown in water (FIG. 6A), GMHA pre-gel solution (FIG. 6B),and GMHA hydrogel (FIG. 6C). Aligned alginate microparticles made asdescribed above are shown in water (FIG. 7A), GMHA pre-gel solution(FIG. 7B), and GMHA hydrogel (FIG. 7C). FIGS. 8A-8C illustrate compositeimages of crosslinked GMHA hydrogels made as described above withunaligned alginate microparticles (FIG. 8A), aligned alginatemicroparticles (FIG. 8B), and aligned and degraded alginatemicroparticles (FIG. 8C). Finally, FIG. 9 Illustrates porous channelsremaining after particle degradation. The channels were imaged underconfocal microscopy after backfilling with Dextran-FITC.

REFERENCES FOR EXAMPLE 1

-   [1] J. S. Belkas, M. S. Shoichet, and R. Midha, “Axonal guidance    channels in peripheral nerve regeneration,” Operative Techniques in    Orthopaedics, vol. 14, pp. 190-198, July 2004.-   [2] S. Ichihara, Y. Inada, and T. Nakamura, “Artificial nerve tubes    and their application for repair of peripheral nerve injury: an    update of current concepts,” Injury, vol. 39, pp. 29-39, October    2008.-   [3] K. Brattain, “Analysis of the Peripheral Nerve Injury Market in    the United States,” Magellan Medical Technology Consultants, Inc.,    Minneapolis, Minn., 2013.-   [4] S. K. Lee and S. W. Wolfe, “Peripheral nerve injury and repair,”    J Am Acad Orthop Surg, vol. 8, pp. 243-52, July-August 2000.-   [5] T. W. Hudson, S. Y. Liu, and C. E. Schmidt, “Engineering an    improved acellular nerve graft via optimized chemical processing,”    Tissue engineering, vol. 10, pp. 1346-1358, 2004.-   [6] T. W. Hudson, S. Zawko, C. Deister, S. Lundy, C. Y. Hu, K. Lee,    and C. E. Schmidt, “Optimized acellular nerve graft is    immunologically tolerated and supports regeneration,” Tissue    engineering, vol. 10, pp. 1641-1651, 2004.-   [7] D. N. Brooks, R. V. Weber, J. D. Chao, B. D. Rinker, J.    Zoldos, M. R. Robichaux, S. B. Ruggeri, K. A. Anderson, E. E.    Bonatz, S. M. Wisotsky, M. S. Cho, C. Wilson, E. O. Cooper, J. V.    Ingari, B. Safa, B. M. Parrett, and G. M. Buncke, “Processed nerve    allografts for peripheral nerve reconstruction: a multicenter study    of utilization and outcomes in sensory, mixed, and motor nerve    reconstructions,” Microsurgery, vol. 32, pp. 1-14, January 2012.-   [8] E. C. Spivey, Z. Z. Khaing, J. B. Shear, and C. E. Schmidt, “The    fundamental role of subcellular topography in peripheral nerve    repair therapies,” Biomaterials, vol. 33, pp. 4264-4276, Jul. 1,    2012.-   [9] S. Kehoe, X. F. Zhang, and D. Boyd, “FDA approved guidance    conduits and wraps for peripheral nerve injury: A review of    materials and efficacy,” Injury, vol. 43, pp. 553-572, Jun. 1, 2012.-   [10] D. Hoffman-Kim, J. A. Mitchel, and R. V. Bellamkonda,    “Topography, Cell Response, and Nerve Regeneration,” Annual Review    of Biomedical Engineering, vol. 12, pp. 203-231, July 2010.-   [11] V. Mukhatyar, L. Karumbaiah, J. Yeh, and R. Bellamkonda,    “Tissue Engineering Strategies Designed to Realize the Endogenous    Regenerative Potential of Peripheral Nerves,” Advanced Materials,    pp. NA-NA, Nov. 10, 2009.-   [12] T. Hadlock, C. Sundback, D. Hunter, M. Cheney, and J. P.    Vacanti, “A polymer foam conduit seeded with Schwann cells promotes    guided peripheral nerve regeneration,” Tissue Eng, vol. 6, pp.    119-27, April 2000.-   [13] S. Stokols and M. H. Tuszynski, “Freeze-dried agarose scaffolds    with uniaxial channels stimulate and guide linear axonal growth    following spinal cord injury,” Biomaterials, vol. 27, pp. 443-451,    February 2006.-   [14] J. B. Scott, M. Afshari, R. Kotek, and J. M. Saul, “The    promotion of axon extension in vitro using polymer-templated fibrin    scaffolds,” Biomaterials, vol. 32, pp. 4830-4839, Jul. 1, 2011.-   [15] K. T. Morin and R. T. Tranquillo, “Guided sprouting from    endothelial spheroids in fibrin gels aligned by magnetic fields and    cell-induced gel compaction,” Biomaterials, vol. 32, pp. 6111-6118,    Sep. 1, 2011.-   [16] N. Dubey, P. C. Letourneau, and R. T. Tranquillo, “Guided    neurite elongation and Schwann cell invasion into magnetically    aligned collagen in simulated peripheral nerve regeneration,”    Experimental neurology, vol. 158, pp. 338-350, 1999.-   [17] D. Ceballos, X. Navarro, N. Dubey, G.    Wendelschafer-Crabb, W. R. Kennedy, and R. T. Tranquillo,    “Magnetically aligned collagen gel filling a collagen nerve guide    improves peripheral nerve regeneration,” Experimental neurology,    vol. 158, pp. 290-300, 1999.-   [18] R. T. Tranquillo, T. S. Girton, B. A. Bromberek, T. G. Triebes,    and D. L. Mooradian, “Magnetically orientated tissue-equivalent    tubes: application to a circumferentially orientated    media-equivalent,” Biomaterials, vol. 17, pp. 349-357, 1996.-   [19] M. R. Sommer, R. M. Erb, and A. R. Studart, “Injectable    Materials with Magnetically Controlled Anisotropic Porosity,” ACS    Applied Materials &amp; Interfaces, vol. 4, pp. 5086-5091, Oct. 24,    2012.-   [20] Y. Li, G. Huang, X. Zhang, B. Li, Y. Chen, T. Lu, T. J. Lu,    and F. Xu, “Magnetic Hydrogels and Their Potential Biomedical    Applications,” Advanced Functional Materials, vol. 23, pp. 660-672,    Sep. 27, 2012.-   [21] C. Hu, C. Tercero, S. Ikeda, M. Nakajima, H. Tajima, Y.    Shen, T. Fukuda, and F. Arai, “Biodegradable porous sheet-like    scaffolds for soft-tissue engineering using a combined particulate    leaching of salt particles and magnetic sugar particles,” JBIOSC,    vol. 116, pp. 126-131, Jul. 1, 2013.-   [22] C. Hu, T. Uchida, C. Tercero, S. Ikeda, K. Ooe, T. Fukuda, F.    Arai, M. Negoro, and G. Kwon, “Development of biodegradable    scaffolds based on magnetically guided assembly of magnetic sugar    particles,” Journal of Biotechnology, vol. 159, pp. 90-98, Jun. 31,    2012.-   [23] B. R. Whatley, X. Li, N. Zhang, and X. Wen, “Magnetic-directed    patterning of cell spheroids,” Journal of Biomedical Materials    Research Part A, vol. 102, pp. 1537-1547, Jul. 2, 2013.-   [24] S. Tasoglu, D. Kavaz, U. A. Gurkan, S. Guven, P. Chen, R.    Zheng, and U. Demirci, “Paramagnetic Levitational Assembly of    Hydrogels,” Advanced Materials, vol. 25, pp. 1137-1143, Dec. 10,    2012.-   [25] L. H. Reddy, J. L. Arias, J. Nicolas, and P. Couvreur,    “Magnetic Nanoparticles: Design and Characterization, Toxicity and    Biocompatibility, Pharmaceutical and Biomedical Applications,”    Chemical Reviews, vol. 112, pp. 5818-5878, Nov. 14, 2012.-   [26] N. Lewinski, V. Colvin, and R. Drezek, “Cytotoxicity of    Nanoparticles,” Small, vol. 4, pp. 26-49, Feb. 18, 2008.-   [27] W. R. Gombotz and S. F. Wee, “Protein release from alginate    matrices,” Advanced Drug Delivery Reviews, vol. 64, pp. 194-205,    2012.-   [28] H. H. Tønnesen and J. Karlsen, “Alginate in drug delivery    systems,” Drug development and industrial pharmacy, vol. 28, pp.    621-630, 2002.-   [29] C. Bucke, “Cell Immobilization in Calcium Alginate,” Methods in    enzymology, vol. 135, pp. 175-189, 1987.-   [30] Z. Z. Khaing and C. E. Schmidt, “Advances in natural    biomaterials for nerve tissue repair,” Neuroscience Letters, vol.    519, pp. 103-114, Jul. 25, 2012.-   [31] S. K. Seidlits, Z. Z. Khaing, R. R. Petersen, J. D.    Nickels, J. E. Vanscoy, J. B. Shear, and C. E. Schmidt, “The effects    of hyaluronic acid hydrogels with tunable mechanical properties on    neural progenitor cell differentiation,” Biomaterials, vol. 31, pp.    3930-3940, Jun. 1, 2010.-   [32] A. P. Herrera, C. Barrera, and C. Rinaldi, “Synthesis and    functionalization of magnetite nanoparticles with aminopropylsilane    and carboxymethyldextran,” Journal of Materials Chemistry, vol.    18, p. 3650, 2008.-   [33] V. L. Calero-DdelC, A. M. Gonzalez, and C. Rinaldi, “A    Statistical Analysis to Control the Growth of Cobalt Ferrite    Nanoparticles Synthesized by the Thermodecomposition Method,”    Journal of Manufacturing Science and Engineering, vol. 132, p.    030914, 2010.-   [34] C. E. Schmidt and J. B. Leach, “Neural tissue engineering:    strategies for repair and regeneration,” Annu Rev Biomed Eng, vol.    5, pp. 293-347, 2003.-   [35] S. Suri and C. E. Schmidt, “Photopatterned collagen-hyaluronic    acid interpenetrating polymer network hydrogels,” Acta Biomater,    vol. 5, pp. 2385-97, September 2009.-   [36] P. Danhier, G. De Preter, S. Boutry, I. Mahieu, P. Leveque, J.    Magat, V. Haufroid, P. Sonveaux, C. Bouzin, O. Feron, R. N.    Muller, B. F. Jordan, and B. Gallez, “Electron paramagnetic    resonance as a sensitive tool to assess the iron oxide content in    cells for MRI cell labeling studies,” Contrast media &amp; molecular    imaging, vol. 7, pp. 302-307, May 26, 2012.-   [37] V. Ayala, A. P. Herrera, M. Latorre-Esteves, M. Torres-Lugo,    and C. Rinaldi, “Effect of surface charge on the colloidal stability    and in vitro uptake of carboxymethyl dextran-coated iron oxide    nanoparticles,” Journal of Nanoparticle Research, vol. 15, p. 1874,    Jul. 30, 2013.-   [38] S. K. Seidlits, C. E. Schmidt, and J. B. Shear,    “High-Resolution Patterning of Hydrogels in Three Dimensions using    Direct-Write Photofabrication for Cell Guidance,” Advanced    Functional Materials, vol. 19, pp. 3543-3551, Nov. 23, 2009.-   [39] P. Dinh, A. Hazel, W. Palispis, S. Suryadevara, and R. Gupta,    “Functional assessment after sciatic nerve injury in a rat model,”    Microsurgery, vol. 29, pp. 644-9, 2009.

Example 2 Three-Dimensionally Templated Hydrogels for Rat Sciatic NerveInjury Repair

The present example describes an embodiment of a method for templatingnatural hydrogels with a linearly-oriented, three-dimensional porousarchitecture, which mimics the architecture of native peripheral nerve.

The present example describes making a bioengineered peripheral nerverepair scaffold including both chemical and architectural components ofthe natural peripheral nerve for repair of long nerve gaps. Hydrogelnerve repair scaffolds were developed using natural extracellular matrixcomponents, with three-dimensional porous architecture similar to nativenerve via templating with linearly-aligned, degradable magneticmicroparticles.

In the present example, glycidyl methacrylated hyaluronic acid hydrogelswere synthesized with 1.5 mg/mL Collagen I incorporated to allow forcell adhesion (GMHA-Col hydrogels). GMHA was dissolved at 2× the desiredfinal concentration in 1% Irgacure 2959 (12959, photocrosslinkinginitiator) and water overnight under agitation. Collagen 1 solution andMAMs were added, and sufficient H2O was added to bring the solution tothe final dilution volume. Ultimately, the pre-gel solution contained 20mg/mL GM HA, 0.3% 12959, 1.5 mg/mL Collagen I, and 6 mg/mL MAMs. Thepre-gel solution was mixed on an asymmetrical mixer (FlackTek) foruniform mixture all components and dispersion of MAMs.

Magnetic alginate microparticles (MAMs) were prepared viaemulsification, using alginic acid and magnetite nanoparticles asdescribed in Example 1, above.

The MAMs were added to the GMHA-Col hydrogel solution prior to gelation.The solution was injected into a silicone mold placed between 2 glassslides, and the mold was placed within a cylindrical magnet (asdescribed in Example 1 above) for 20 minutes for particle alignment.After alignment, the molds were removed from the magnet and immediatelyplaced under UV light for 10 minutes to crosslink the GMHA, followed by40 minutes of incubation at 37° C. to induce collagen fibrillogenesis.Magnetic alginate microparticles were dissolved using alternating washesin 0.1M EDTA and 2 unit/mL alginate lyase for 12 days at 37° C. (threecycles of 1 day alginate lyase, 3 days EDTA). In other experiments, EDTAwashes were shortened to 2 days each (total 9 days), which successfullydissolved the particles (data not shown). Finally, the hydrogels wereequilibrated in DMEM base culture media for 1 week to remove residualEDTA and alginate-lyase.

Resulting templated hydrogels were wrapped with decellularized,small-intestinal submucosa (SIS) to create hydrogel implants capable ofbeing sutured. In vivo sciatic nerve implants were conducted in Lewisrats using 3 experimental groups: isograft fresh-nerve repair, templatedGMHA-Col hydrogels made as described above, and non-templated GMHA-Colhydrogels with identical hydrogel composition to the templated groupexcept without the addition/dissolution of magnetic alginatemicroparticles. For each group, 8 mm of sciatic nerve was removed tocreate a 10 mm nerve gap with tension-free repair after implantation ofthe experimental device. Implants were harvested at 2 and 4-weekendpoints (n=3 per group at each endpoint), for sectioning andimmunohistochemical analysis.

The present example demonstrates successful templating of GMHA-Colhydrogels using aligned magnetic particles. Particle alignment ofdensely-packed particle chains with millimeter-scale alignment lengthand tunable diameters of 10-100 μm was achieved. The particles weresuccessfully degraded from crosslinked hydrogels, leaving behind porouschannels. All rats survived implantation surgeries and recoveredsuccessfully through to the designated endpoints. Preliminary analysisof longitudinal sections clearly demonstrates greater cellularinfiltration and hydrogel remodeling in templated GMHA-Col hydrogels vs.non-templated controls after 2 weeks of implantation (FIGS. 10A and10B). Furthermore, templated hydrogel implants appear to besignificantly degraded at 2 weeks, as compared to non-templatedhydrogels that are primarily intact. This earlier degradation isdesirable since with the formation of a fibrin cable is not necessarywith the templated hydrogel implants of the present disclosure, whichallows more rapid remodeling of nerve fibers.

1-32. (canceled)
 33. A method of making a biocompatible, tissue scaffoldhaving aligned microchannels, the method comprising: providing abiocompatible precursor material capable of polymerizing or crosslinkingto form a gel or solid material upon activation; providingmicroparticles comprising one or more magnetic nanoparticlesencapsulated in a dissolvable, biocompatible matrix material; combiningthe microparticles and the biocompatible liquid material in a mold;applying a magnetic field to the combined microparticles andbiocompatible liquid such that the microparticles spatially align withinthe biocompatible material forming a plurality of columns of adjacentmicroparticles, where the columns are substantially directionallyaligned with one another; activating the biocompatible precursormaterial to crosslink or polymerize such that the biocompatible materialsubstantially solidifies, forming a three dimensional (3D) scaffoldaround the aligned microparticles; dissolving the matrix material of themicroparticles to produce a plurality of aligned voids and microchannelswithin the scaffold; and allowing the dissolved matrix material and thereleased magnetic nanoparticles to diffuse out of the aligned voids andmicrochannels within the scaffold.
 34. The method of claim 33, whereinthe biocompatible scaffold is biodegradable.
 35. The method of claim 33,wherein a portion of the microchannels or a network of interconnectedmicrochannels, or both, extend the length of the scaffold.
 36. Themethod of claim 33, wherein the scaffold is about 2 to 12 cm in length.37. The method of claim 33, wherein the microparticles have a diameterof about 100 nm to 100 μm.
 38. The method of claim 33, wherein themicrochannels have a diameter of about 1 to 100 μm
 39. The method ofclaim 33, wherein the biocompatible precursor material comprises ahydrogel precursor that forms a hydrogel upon crosslinking.
 40. Themethod of claim 33, wherein the biocompatible precursor materialcomprises hyaluronic acid and collagen, wherein the material crosslinksto form a hyaluronic acid/collagen hydrogel upon application ofultraviolet light.
 41. The method of claim 33, wherein the magneticnanoparticles comprise iron oxide nanoparticles
 42. The method of claim33, wherein the microparticle matrix material comprises a biocompatible,dissolvable hydrogel.
 43. The method of claim 33, wherein themicroparticles comprise iron oxide nanoparticles encapsulated in abiocompatible, dissolvable, alginate hydrogel matrix.
 44. The method ofclaim 43, wherein the microparticles are prepared by combining awater-in-oil emulsion of an alginate salt and iron oxide nanoparticlemixture and crosslinking with a calcium salt to form microparticlescomprising iron oxide nanoparticles encapsulated in a crosslinkedalginate hydrogel matrix.
 45. The method of claim 33, wherein thebiocompatible precursor material, the microparticle matrix material, orboth further comprises cells, biomolecules, or both.
 46. A biocompatibletissue scaffold comprising: a three-dimensional (3D) biocompatiblescaffold material; and a plurality of magnetically templated,substantially aligned microchannels having a diameter of about 1 toabout 100 μm, wherein a portion of the microchannels, a network ofinterconnected microchannels, or both extend the length of the scaffold.47. The biocompatible, tissue scaffold of claim 46, wherein thebiocompatible scaffold material is biodegradable.
 48. The biocompatible,tissue scaffold of claim 47, wherein the biocompatible scaffold materialcomprises a hyaluronic acid hydrogel and collagen.
 49. Thebiocompatible, tissue scaffold of claim 46, wherein a plurality of themicrochannels have a diameter of about 5 to about 20 μm.
 50. Thebiocompatible, tissue scaffold of claim 46, made by steps comprising:providing a biocompatible precursor material capable of polymerizing orcrosslinking to form a gel or solid material upon activation; providingmicroparticles comprising one or more magnetic nanoparticlesencapsulated in a dissolvable, biocompatible matrix material; combiningthe microparticles and the biocompatible liquid material in a mold;applying a magnetic field to the combined microparticles andbiocompatible liquid such that the microparticles spatially align withinthe biocompatible material forming a plurality of columns of adjacentmicroparticles, where the columns are substantially directionallyaligned with one another; activating the biocompatible precursormaterial to crosslink or polymerize such that the biocompatible materialsubstantially solidifies, forming the three dimensional (3D) scaffoldaround the aligned microparticles; dissolving the matrix material of themicroparticles to produce the plurality of magnetically templated,substantially aligned microchannels within the scaffold; and allowingthe dissolved matrix material and the released magnetic nanoparticles todiffuse out of the aligned voids and microchannels within the scaffold.51. A method of repairing peripheral nerve damage, the methodcomprising: repairing a peripheral nerve gap with a biocompatible,magnetically templated tissue scaffold comprising: a three-dimensional(3D) biocompatible scaffold material, wherein the 3D biocompatiblescaffold material optionally includes cells, biomolecules, or both; anda plurality of magnetically templated, aligned microchannels having adiameter of about 1 to about 100 μm, wherein a portion of themicrochannels or a network of interconnected microchannels, or bothextend the length of the scaffold and wherein a portion of themagnetically templated microchannels optionally include cells,biomolecules, or both.
 52. The method of claim 51, wherein the nerve gapis about 2-12 cm in length.